U.S. Pat. No. 4,046,636 disclosed enzyme conjugates of benzodiazepines useful in a homogeneous enzyme immunoassay for benzodiazepines such as oxazepam, and diazepam and chloridazepoxide. In Example 8 of this patent there is disclosed the preparation of a conjugate from N-desmethyldiazepam and bovine gamma globulin through a 3'-carboxypropyl linking group attached to the 1-position of the benzodiazepine nucleus. This material is used as an immunogen to elicit antibodies which cross-react strongly with oxazepam, diazepam and chlordiazepoxide, as well as the metabolite N-desmethyldiazepam.
U.S. Pat. Application Ser. No. 784,101 filed Apr. 4, 1977 discloses a radioimmunoassay for benzodiazepines such as diazepam, chlordiazepoxide, oxazepam, and metabolites thereof. The assay employs both known and novel antibodies. Novel antibodies are elicited using the diazonium salt of the 5-(4-aminophenyl)benzodiazepine hapten coupled to the immunogenic carrier material as the immunogen. The radioligands employed in this assay are novel .sup.125 I-labelled 4'-hydroxy derivatives of the compounds to be detected.
Peskar and Spector described a radioimmunoassay procedure useful in detecting nanogram amounts of diazepam or N-desmethyldiazepam in plasma samples in J. Pharmacol Exp. Ther. 186, 167 (1973). This assay utilized .sup.14 C-diazepam as the tracer. Antibodies were elicited using either 5-[3-(4-aminophenylazo)-4-hydroxy-phenyl]-7-chloro-1,3-dihydro-1-methyl-2H -1,4-benzodiazepin-2-one or 7-amino-5-(2-chlorophenyl)-3H-1,4-benzodiazepin-2(1H)-one as haptens which were subsequently coupled to bovine serum albumin (BSA) to form the desired immunogens.
Dixon et al. reported in J. Pharm. Sci. 64, 937 (1975) of a radioimmunoassay for chlordiazepoxide in plasma. Once again the tracer compound was a .sup.14 C-labelled compound. The immunogen was derived by coupling the reactive acyl azide of 7-chloro-5-(4-hydrazinocarbonylmethoxyphenyl)-2-methylamino-3H-1,4-benzodi azepine-4-oxide to BSA.
Spin labelling of benzodiazepines for use in a Free Radical Assay Technique is disclosed by Goldstein et al. in U.S. Pat. No. 3,690,834. Specific benzodiazepines disclosed include chlordiazepoxide, diazepam and oxazepam.
Another paper by Dixon et al., J. Pharm. Sci. 66 235 (1977) describes a radioimmunoassay for clonazepam using an immunogen consisting of 3-hemisuccinoyloxyclonazepam covalently bound to BSA. The tracer employed for the assay was .sup.3 H-clonazepam.